The long-term goal of this project is to explore the mechanisms and molecular ordering of radiation-induced apoptosis in mammalian cells and the anti-apoptotic signaling systems that constrain this response. The basic hypothesis is that failure to initiate apoptosis in response to radiation results from tight regulation of the apoptotic response by anti-apoptotic mechanisms. The balance between pro- and anti-apoptotic systems determines under any set of circumstances the magnitude of the apoptotic response in vitro and in vivo. While the p53 pathway is the most acclaimed mechanism of radiation-induced apoptosis, we have studied in previous rounds of this research the mechanisms and molecular ordering of ceramide-mediated apoptosis, specifically of the ceramide generated by acid sphingomyelinase activation in response to radiation in vitro and in vivo. Our recent discovery that radiation-induced DNA damage signals apoptosis via activating another enzyme, ceramide synthase (CS), and the identification of the mammalian CS gene, provide new opportunities for mechanistic studies of radiation sensitivity and resistance in normal and tumor tissues. This proposal presents a detailed plan to characterize mammalian CS and the effects of radiation on its structure and function. Specifically, we propose to purify mammalian CS, develop an in vitro assay for its enzymatic activity, characterize its molecular structure, identify post-translational modifications on CS induced by radiation, and explore the mechanisms of ATM-mediated suppression of radiation-induced CS activation. We will also study the mechanisms of CS involvement in mitochondrial apoptosis, focusing on demonstrating a requirement for CS activation in radiation-induced mitochondrial apoptosis, the topology of CS in the mitochondrial membrane, ceramide species generated by CS after irradiation, the interaction between ceramide and BAX, and the effects of CS activation on the MPT complex. Finally we propose to study the involvement of CS in tissue responses to radiation in vivo, specifically focusing on the involvement of CS in the GI tract and tumor responses to single-dose and fractionated irradiation. The proposed Specific Aims are interactive and address new and heretofore unknown mechanisms of radiation-induced cell death and tissue responses. The research plan provides an approach to investigate new hypotheses on the mechanisms of radiation resistance. Improved understanding of these pro- and anti-apoptotic signaling systems and their coordinated function may yield opportunities for pharmacological interventions in in vivo models, with important potentials for clinical applications in the radiation management of human cancer.